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Fibrosis is regulated by interactions between immune and mesenchymal cells. However, the capacity of cell types to modulate human fibrosis pathology is poorly understood due to lack of a fully humanized model system. MISTRG6 mice were engineered by homologous mouse/human gene replacement to develop an immune system like humans when engrafted with human hematopoietic stem cells (HSCs). We utilized MISTRG6 mice to model scleroderma by transplantation of healthy or scleroderma skin from a patient with pansclerotic morphea to humanized mice engrafted with unmatched allogeneic HSC. We identified that scleroderma skin grafts contained both skin and bone marrow-derived human CD4 and CD8 T cells along with human endothelial cells and pericytes. Unlike healthy skin, fibroblasts in scleroderma skin were depleted and replaced by mouse fibroblasts. Furthermore, HSC engraftment alleviated multiple signatures of fibrosis, including expression of collagen and interferon genes, and proliferation and activation of human T cells. Fibrosis improvement correlated with reduced markers of T cell activation and expression of human IL-6 by mesenchymal cells. Mechanistic studies supported a model whereby IL-6 trans-signaling driven by CD4 T cell-derived soluble IL-6 receptor complexed with fibroblast-derived IL-6 promoted excess extracellular matrix gene expression. Thus, MISTRG6 mice transplanted with scleroderma skin demonstrated multiple fibrotic responses centered around human IL-6 signaling, which was improved by the presence of healthy bone marrow-derived immune cells. Our results highlight the importance of IL-6 trans-signaling in pathogenesis of scleroderma and the ability of healthy bone marrow-derived immune cells to mitigate disease.
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Basidiomycota , Esclerodermia Localizada , Humanos , Animales , Ratones , Interleucina-6 , Células Endoteliales , Piel , Modelos Animales de EnfermedadRESUMEN
PURPOSE OF REVIEW: Scleroderma and other fibrotic diseases have been investigated using single-cell RNA sequencing (scRNA-Seq), which has demonstrated enrichment in myeloid cell populations in multiple tissues. However, scRNA-Seq studies are inconsistent in their nomenclature of myeloid cell types, including dendritic cells, monocytes, and macrophages. Using cell type-defining gene signatures, I propose a unified nomenclature through analysis of myeloid cell enrichment across fibrotic tissues. RECENT FINDINGS: scRNA-Seq of human blood and skin identified a new subset of dendritic cells called DC3. DC3 express similar inflammatory genes to monocytes, including FCN1, IL1B, VCAN, S100A8, S100A9, and S100A12. DC3 can be distinguished from monocytes by expression of EREG and Fc receptor genes such as FCER1A and FCGR2B. scRNA-Seq analyses of scleroderma skin and lung, idiopathic pulmonary fibrosis (IPF), COVID-19 lung fibrosis, myelofibrosis, and liver, kidney, and cardiac fibrosis all showed enrichment in myeloid cell types. Although they were called different names, studies of scleroderma skin and lung as well as liver cirrhosis datasets demonstrated enrichment in DC3. By contrast, lung, heart, and kidney fibrosis were enriched in SPP1 macrophages. High numbers of DC3 in the skin was associated with worse SSc skin and lung fibrosis severity. SUMMARY: scRNA-Seq of multiple diseases showed enrichment of DC3 in fibrotic skin, lung, and liver, whereas SPP1 macrophages occurred in fibrotic lung, heart, and kidney. Because DC3 and SPP1 macrophages showed organ-specific enrichment, understanding their signaling mechanisms across tissues will be important for future investigation.
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Single-cell RNA sequencing has been widely used to investigate cell state transitions and gene dynamics of biological processes. Current strategies to infer the sequential dynamics of genes in a process typically rely on constructing cell pseudotime through cell trajectory inference. However, the presence of concurrent gene processes in the same group of cells and technical noise can obscure the true progression of the processes studied. To address this challenge, we present GeneTrajectory, an approach that identifies trajectories of genes rather than trajectories of cells. Specifically, optimal transport distances are calculated between gene distributions across the cell-cell graph to extract gene programs and define their gene pseudotemporal order. Here we demonstrate that GeneTrajectory accurately extracts progressive gene dynamics in myeloid lineage maturation. Moreover, we show that GeneTrajectory deconvolves key gene programs underlying mouse skin hair follicle dermal condensate differentiation that could not be resolved by cell trajectory approaches. GeneTrajectory facilitates the discovery of gene programs that control the changes and activities of biological processes.
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Most of the DNA in eukaryotes is packaged in tandemly arrayed nucleosomes that, together with numerous DNA- and nucleosome-associated enzymes and regulatory factors, make up chromatin. Chromatin modifying and remodeling agents help regulate access to selected DNA segments in chromatin, thereby facilitating transcription and DNA replication and repair. Studies of nucleotide excision repair (NER), single strand break repair (SSBR), and the homology-directed repair (HDR), and non-homologous end-joining (NHEJ) double strand break repair pathways have led to an "access-repair-restore" paradigm, in which chromatin in the vicinity of damaged DNA is disrupted, thereby enabling efficient repair and the subsequent repackaging of DNA into nucleosomes. When damage is extensive, these repair processes are accompanied by cell cycle checkpoint activation, which provides cells with sufficient time to either complete the repair or initiate apoptosis. It is not clear, however, if base excision repair (BER) of the ~20,000 or more oxidative DNA damages that occur daily in each nucleated human cell can be viewed through this same lens. Until recently, we did not know if BER requires or is accompanied by nucleosome disruption, and it is not yet clear that anything short of overwhelming oxidative damage (resulting in the shunting of DNA substrates into other repair pathways) results in checkpoint activation. This review highlights studies of how oxidatively damaged DNA in nucleosomes is discovered and repaired, and offers a working model of events associated with BER in chromatin that we hope will have heuristic value.
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Cromatina/metabolismo , Daño del ADN , Reparación del ADN , Animales , ADN Glicosilasas/metabolismo , ADN Ligasas/metabolismo , ADN Polimerasa beta/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Ratones , Nucleosomas/metabolismo , Nucleótidos/química , Estrés Oxidativo/fisiología , Transducción de SeñalRESUMEN
Immune cells are fundamental regulators of extracellular matrix (ECM) production by fibroblasts and have important roles in determining extent of fibrosis in response to inflammation. Although much is known about fibroblast signaling in fibrosis, the molecular signals between immune cells and fibroblasts that drive its persistence are poorly understood. We therefore analyzed skin and lung samples of patients with diffuse cutaneous systemic sclerosis, an autoimmune disease that causes debilitating fibrosis of the skin and internal organs. Here, we define a critical role of epiregulin-EGFR signaling between dendritic cells and fibroblasts to maintain elevated ECM production and accumulation in fibrotic tissue. We found that epiregulin expression marks an inducible state of DC3 dendritic cells triggered by type I interferon and that DC3-derived epiregulin activates EGFR on fibroblasts, driving a positive feedback loop through NOTCH signaling. In mouse models of skin and lung fibrosis, epiregulin was essential for persistence of fibrosis in both tissues, which could be abrogated by epiregulin genetic deficiency or a neutralizing antibody. Therapeutic administration of epiregulin antibody reversed fibrosis in patient skin and lung explants, identifying it as a previously unexplored biologic drug target. Our findings reveal epiregulin as a crucial immune signal that maintains skin and lung fibrosis in multiple diseases and represents a promising antifibrotic target.
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Fibrosis Pulmonar , Ratones , Animales , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Ligandos , Piel/patología , Fibrosis , Células DendríticasRESUMEN
Sarcoidosis is an idiopathic inflammatory disorder that is commonly treated with glucocorticoids. An imprecise understanding of the immunologic changes underlying sarcoidosis has limited therapeutic progress. Here in this open-label trial (NCT03910543), 10 patients with cutaneous sarcoidosis are treated with tofacitinib, a Janus kinase inhibitor. The primary outcome is the change in the cutaneous sarcoidosis activity and morphology instrument (CSAMI) activity score after 6 months of treatment. Secondary outcomes included change in internal organ involvement, molecular parameters, and safety. All patients experience improvement in their skin with 6 patients showing a complete response. Improvement in internal organ involvement is also observed. CD4+ T cell-derived IFN-γ is identified as a central cytokine mediator of macrophage activation in sarcoidosis. Additional type 1 cytokines produced by distinct cell types, including IL-6, IL-12, IL-15 and GM-CSF, also associate with pathogenesis. Suppression of the activity of these cytokines, especially IFN-γ, correlates with clinical improvement. Our results thus show that tofacitinib treatment is associated with improved sarcoidosis symptoms, and predominantly acts by inhibiting type 1 immunity.
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Pirimidinas , Sarcoidosis , Citocinas/metabolismo , Humanos , Piperidinas/farmacología , Piperidinas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Sarcoidosis/tratamiento farmacológico , Sarcoidosis/patologíaRESUMEN
Morphea, also known as localized scleroderma, is characterized by inflammation and fibrosis of the skin. The exact pathogenesis of morphea is unknown, but generally includes genetic predisposition to autoimmunity combined with an environmental insult. Previous cases have been associated with active Borrelia infection; however, Borrelia infection as a direct cause of morphea was not generalizable to most patients. Within endemic areas, Borrelia burgdorferi is the most common cause of facial nerve paralysis, another autoimmune phenomenon. We report a case of facial morphea in a young man with family history of autoimmune disease who developed morphea in the same location as two previous episodes of Borrelia-induced facial nerve palsy. This case is remarkable because it suggests Borrelia burgdorferi induced loss of local immune tolerance to host antigens, first with facial nerve palsy and followed years later by development of morphea.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Polymerase chain reaction (PCR) amplification of the heat shock protein 65 (hsp65) gene followed by high-resolution melting analysis with LCGreen I (Idaho Technology, Salt Lake City, UT) was used to differentiate the mycobacteria species Mycobacterium chelonae, Mycobacterium abscessus, and Mycobacterium immunogenum in less than 20 minutes. A 105-base-pair amplicon that clustered the different species by predicted melting temperature was found from available GenBank hsp65 sequences. We identified 24 clinical isolates within the M chelonae-abscessus group by proximal 16S ribosomal RNA and hsp65 gene sequencing. Rapid-cycle PCR followed by high-resolution melting analysis clustered these samples into the following groups: M abscessus, 12; M abscessus sequence variant, 2; M chelonae, 7; unexpected M chelonae sequence variant, 1; and M immunogenum, 2. The M chelonae variant had a single base change not found in reported GenBank sequences. Advantages of the method include speed, low risk of amplicon contamination (closed-tube), and no need for separation steps (sequencing, electrophoresis, high-performance liquid chromatography) or real-time monitoring.
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Proteínas Bacterianas/genética , Chaperoninas/genética , Mycobacterium chelonae/clasificación , Micobacterias no Tuberculosas/clasificación , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Chaperonina 60 , Datos de Secuencia Molecular , Mycobacterium chelonae/genética , Micobacterias no Tuberculosas/genética , FilogeniaRESUMEN
IMPORTANCE: Inhibition of angiogenesis is an effective anticancer strategy because neoplasms require a rich blood supply. Ramucirumab, approved by the US Food and Drug Administration in 2014 to treat gastric adenocarcinomas and non-small cell lung carcinomas, targets vascular endothelial growth factor 2 (VEGFR2). We identified a patient prescribed a regimen of irinotecan hydrochloride, cetuximab, and ramucirumab for metastatic rectal cancer (diagnosed in November 2013 and treated through early January 2015) who developed a new-onset, expanding vascular lesion on his right leg. Via exome sequencing, we found that the lesion contained a single somatic mutation in KDR (encodes VEGFR2), possibly in response to ramucirumab. Vascular tumors are not a known complication of antiangiogenic therapeutics. OBSERVATIONS: Exome sequencing of the well-demarcated, blanching vascular lesion on the lateral right shin revealed a somatic p.T771R mutation in KDR, without evidence of other somatic mutations or loss of heterozygosity. Histological features included lobules of small vessels within the dermis, resembling a tufted angioma. CONCLUSIONS AND RELEVANCE: A potential adverse effect of ramucirumab in combination therapy is the development of sporadic angiomas. The p.T771R mutation was previously implicated in autophosphorylation of VEGFR2 and reported in angiosarcomas alongside other driver mutations. Our observations suggest that this mutation confers a proliferative advantage in the setting of ramucirumab therapy. Patients receiving ramucirumab should be monitored for the development of new vascular lesions.
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Anticuerpos Monoclonales/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Hemangioma/inducido químicamente , Neoplasias Cutáneas/inducido químicamente , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Adulto , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/efectos adversos , Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Cetuximab/administración & dosificación , Hemangioma/patología , Humanos , Irinotecán , Masculino , Mutación , Neoplasias del Recto/tratamiento farmacológico , Neoplasias del Recto/patología , Neoplasias Cutáneas/patología , RamucirumabRESUMEN
Immunofluorescence is a laboratory technique that utilizes a fluorophore-labeled antibody to detect immune complexes in tissue. Most of the labeled antibodies used in a clinical laboratory bind the conserved domains within each class of human antibodies, allowing them to detect a wide range of autoimmune complexes. Drawbacks to this technique mostly relate to proper handling of the specimen and the fluorophore-labeled antibodies. Therefore, having a basic understanding of fluorophores and antibodies is important for processing a specimen that yields a high signal-to-background ratio as well as troubleshooting problems, should they arise.